Search

The Gs-protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a novel chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the new A2AAR crystallization construct devoid of ligand binding site mutations.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2–3 form the ligand-binding site, while the membrane proximal domains D4–7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4–5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions. Allosteric inhibition of VEGFR-2 signaling represents an attractive option for the treatment of neovascular diseases. We showed earlier that DARPin® binders to domains D4 or D7 are potent VEGFR-2 inhibitors. Here we investigated in detail the allosteric inhibition mechanism of the domain D4 binding inhibitor D4b. The 2.38 A crystal structure of D4b in complex with VEGFR-2 D4–5, the first high-resolution structure of this VEGFR-2 segment, indicates steric hindrance by D4b as the mechanism of inhibition of receptor activation. At the cellular level, D4b triggered quantitative internalization of VEGFR-2 in the absence of ligand and thus clearance of VEGFR-2 from the surface of endothelial cells. The allosteric VEGFR-2 inhibition was sufficiently strong to efficiently inhibit the growth of human endothelial cells at suboptimal dose in a mouse xenograft model in vivo, underlining the therapeutic potential of the approach.

Mutation of both the intracellular catalytic domain and the extracellular domain of the receptor for epidermal growth factor (EGFR) can drive oncogenicity. Despite clinical success with targeting EGFR catalytic site mutations, no drugs have proven effective in patients expressing allosteric extracellular domain EGFR mutations, including glioblastomas (GBM) where these mutations are highly expressed. We define the molecular mechanism for oncogenic activation of families of extracellular EGFR mutations and reveal how this mechanism renders current generation small molecule ATP-site inhibitors ineffective. We demonstrate that a group of commonly expressed extracellular domain EGFR mutants expressed in GBM is activated by disulfide-bond mediated covalent dimerization, collectively referred to as locked dimerization (LoDi) EGFR oncogenes. Strikingly, small molecules binding to the active kinase conformation (Type I), but not those binding to the inactive kinase conformation (Type II), potently inhibit catalytic site mutants, but induce covalent dimerization and activate LoDi-EGFR oncogenes, manifesting in paradoxical acceleration of proliferation.SignificanceOur data demonstrate how the locked-dimer mechanism of EGFR oncogenesis has a profound impact on the activity of small molecule inhibitors. This provides a mechanistic understanding for the failure of current generation EGFR inhibitors to effectively treat LoDi-EGFR mutants in GBM, and sets guidelines for discovery of selective LoDi-EGFR inhibitors.

We investigated the role of copy number alterations to refine risk stratification in adult Philadelphia chromosome positive (Ph)+ ALL treated with tyrosine kinase inhibitors (TKI) and allogeneic stem cell transplantation (aSCT). 97 Ph+ ALL patients (median age 41 years, range 18-64 years) within the prospective multicenter GMALL studies 06/99 (n=8) and 07/2003 (n=89) were analysed. All patients received TKI and aSCT in first complete remission (CR1). Copy number analysis was performed with SNP arrays and validated by multiplex ligation-dependent probe amplification (MLPA). The frequencies of recurrently deleted genes were: IKZF1, 76%, CDKN2A/2B, 45%, PAX5, 43%, BTG1, 18%, EBF1, 13%, ETV6, 5%, RB, 14%. In univariate analyses, the presence of CDKN2A/2B deletions had a negative impact on all endpoints: overall survival (p=0.023), disease free survival (p=0.012) and remission duration (p=0.036). The negative predictive value of CDKN2A/2B deletions was retained in multivariable analysis along with other factors such as timing of TKI therapy, intensity of conditioning, achieving remission after induction phase I and BTG1 deletions. We therefore conclude that acquired genomic CDKN2A/2B deletions identify a subgroup of Ph+ ALL patients, who have an inferior prognosis despite aSCT in CR1. Their poor outcome was attributable primarily to a high relapse rate after aSCT.

Background: The overall goal of the project ‘StiEL’ is to contribute to the professional development of teachers and other educational staff working at German secondary schools. The aim is to develop an evidence-based training concept for the inclusion of students with diverse abilities. The project is organized as a collaborative research effort of three partnering institutions and funded by the German Federal Ministry of Education and Research from 2018-2021. Methods: To support the on-going transition towards inclusive school practices, a multi-stage approach is envisaged. The first phase aims at a scoping review of existing literature and programmes on inclusion. The overview is supplemented by interviews with school staff members. Training modules are developed in the second project phase. The third phase of StiEL puts the newly developed training program into practice. The knowledge and skills acquired by the participants through the training as well as the teaching and management of inclusive classrooms after the training are evaluated through longitudinal and ethnographic approaches. The final project phase creates a best practice manual and makes the modules available via open access databases. Results: The presentation will focus on the first phase and try to explore the health-related consequences of the transition towards an inclusive school system in Germany for different participants. We will present preliminary results of expert interviews as well as some results from the literature screening. Due to our findings the current practice on German schools towards the road to inclusion is very stressful for all participants. We will explore recommendations for health promoting schools under conditions of inclusion. Conclusions: In terms of health-related consequences for all participants, the road to inclusion is very ambitious but also very stressful. Regarding the development of an inclusive school system, we need to focus much more on health and health promotion.

We investigated the role of copy number alterations to refine risk stratification in adult Philadelphia chromosome positive (Ph)

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1–3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4–7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.

Five realistic tabletop scenarios were designed to facilitate threat preparedness training of Medical, Public Health, Nursing, Emergency Services, Mental Health, Allied Health, and Pharmacy personnel. Training scenarios were (1) student contaminates lettuce (Act) in a state university with Shigella sonnei (Agent), (2) dismissed athlete contaminates ice (Act) at the basketball tournament with Escherichia coli (Agent), (3) workers fail to report abandoned backpacks (Act) at a state fair that contain smallpox virus (Agent), (4) terrorists expose county residents (Act) to Pneumonic plague bacterium (Agent), and (5) infected birds expose field-trip participants (Act) to Avian influenza virus (Agent). Evaluation of the tabletops yielded positive ratings of educational outcomes in these domains: well-structured, organized, plausible, realistic, engaging, on-target, useful, and multidisciplinary. Attendees with previous blended-learning courses on bioterrorism and threat preparedness enhanced performance in the tabletop exercises. Evaluative data indicated a new level of competence and self-confidence about being part of a coordinated, local-level, interdisciplinary response.

Moesin is a member of the ERM (ezrin, radixin, moesin) family of cytoskeleton/membrane structure organizing and signal transduction proteins. Previously, we found an increased expression of moesin during HIV-1 infection. Moesin was also reported to be incorporated into HIV-1 virions. To analyze whether moesin is a host factor affecting the replication cycle of human immunodeficiency virus type 1 (HIV-1), we used small interfering RNAs (siRNAs) to evaluate the effect of moesin knockdown on HIV-1 replication in P4-CCR5 cells. Moesin's knockdown did not affect the cell viability or cell phenotype. Interestingly, we observed a marked increase in viral replication, as demonstrated by enhanced HIV-1 RNA, p24 antigen, and s-galactosidase reporter expression. Moesin-dependent enhancement of HIV-1 replication was confirmed in lymphocytic host cells (Jurkat). These results suggest an overall rather restrictive role of moesin for HIV-1 replication in host cells in vitro.

SummaryThe cytoplasmic domains of ClC chloride channels and transporters are ubiquitously found in eukaryotic family members and have been suggested to be involved in the regulation of ion transport. All cytoplasmic ClC domains share a conserved scaffold that contains a pair of CBS motifs. Here we describe the structure of the cytoplasmic component of the human chloride channel ClC-Ka at 1.6 Å resolution. The structure reveals a dimeric organization of the domain that is unusual for CBS motif containing proteins. Using a biochemical approach combining mutagenesis, crosslinking, and analytical ultracentrifugation, we demonstrate that the interaction interface is preserved in solution and that the distantly related channel ClC-0 likely exhibits a similar structural organization. Our results reveal a conserved interaction interface that relates the cytoplasmic domains of ClC proteins and establish a structural relationship that is likely general for this important family of transport proteins.

Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) is traditionally considered the subtype with the worst prognosis, despite recent improvements in long-term survival brought about by the use of tyrosine kinase inhibitors (TKI) such as imatinib or dasatinib. Allogeneic stem cell transplantation (aSCT) remains the most effective curative post-remission therapy in adults but appears to be less critical in children, indicating a substantial clinical and biological heterogeneity within the subgroup of Ph+ ALL. The ability to segregate Ph+ ALL into subgroups with different prognosis on the basis of reductions of BCR-ABL1 transcript levels during therapy lends further support to the heterogeneity of this type of leukemia, for which the genetic basis is not known. Microarray-based genome-wide profiling studies conducted predominantly in pediatric ALL patients have recently revealed novel recurrent submicroscopic aberrations of genes involved in B-cell development and cell cycle regulation, such as CDKN2A/B, IKZF1, PAX5, ETV6, RB1, BTG1 and EBF1. Deletions of IKZF1, CDKN2A/B and PAX genes have received the most attention due to their high frequency particularly in BCR-ABL1-positive ALL and their association with an inferior prognosis in the setting of combined TKI and chemotherapy. Their prognostic relevance in the setting of allogeneic SCT for adult or pediatric high risk BCP-ALL is not known. We therefore examined whether the negative prognostic role of IKZF1 aberrations and other frequent microdeletions of genes associated with B-cell development can be overcome by allogeneic SCT in CR1. A total of 137 newly diagnosed Ph+ ALL pts. (median age 42 years, range 18-64y, 79 male 58 female) treated within the prospective multicenter GMALL study 07/03 were analyzed. 96 of these patients underwent aSCT in first complete remission (CR), 8 pts. were primary refractory, 12 CR pts. did not undergo aSCT and relapsed, 11 pts. died during induction. Genome-wide copy number analysis in search for acquired copy number alterations (CNA) was performed with Affymetrix SNP 6.0 arrays with anonymous references. Copy number polymorphisms were excluded from the data by comparison with known copy number polymorphisms registered in the UCSC genome browser http://genome.ucsc.edu/, (hg-18). Putatively acquired CNAs were validated by multiplex ligation-dependent probe amplification (MLPA) and germline matched SNP array analysis of n=20 samples within the study. Of the 96 pts. transplanted in CR1, 48 remain in CR (CCR), 30 pts. relapsed after aSCT and 7 died of treatment related causes, survival data only are available for one patient. CDKN2A/B genomic alterations were identified in 41% (40/97) of patients, deletions of IKZF1 and PAX5 were observed in 61% (59/97) and 39% (38/97) of pts., respectively. Univariate analysis of the complete cohort revealed that deletion of CDKN2A/B was the only aberration with a statistically significant negative effect on overall survival (OS) (p=0.003). Among patients transplanted in CR1, IKZF1-deletions were associated with inferior median time to relapse after SCT (56 mos vs. n.r., p=0.01), DFS from SCT (15.6 mos. vs. n.r.; p=0.024) and OS (median 40 mos. vs. not reached (n.r.) p=0.04) compared with the IKZF1 wildtype cohort. Similarly, the prognosis of pts. with CDKN2A/B deletions was inferior in terms of DFS (median 10.6 mos. vs. n.r.; p=0.022) and OS (median 25 mos. vs. n.r.; p=0.01), but not of remission duration from SCT. PAX5 (p=0.07) but not the combination of all three lesions (p=0.14) showed a trend to a worse prognosis. Of the more uncommon genetic aberrations BTLA, EBF1, ETV6, RB1 and BTG1, only the latter was associated with a lower probability of remaining in CR (0% vs. 67% at 5 years; p=0.012) or DFS (0% vs. 52% at 5 years; p=0.043), with a trend towards shorter OS (median 35 mos. vs. 87 mos; p=0.078). In conclusion, genomic lesions of IKZF1, CDKN2 and PAX5 identify a subgroup of Ph+ ALL pts. who have an inferior survival despite undergoing aSCT in CR1. Their poor outcome is attributable primarily to a high relapse rate after SCT, emphasizing the need to introduce additional treatment elements prior to and after aSCT. Disclosures: No relevant conflicts of interest to declare.

Abstract 2535 Background: Minimal residual disease is well established as an important prognostic parameter in a number of hematologic diseases and is used for stratification and treatment decisions in adult and childhood acute lymphoblastic leukaemia (ALL). DNA- based PCR analysis of Ig-/TCR-rearrangements have been well standardised during the last decade, resulting in robust systems for MRD analysis with an intra- and inter-assay variability of less than half a log and low false positivity. In Ph+ALL, MRD analysis relies on RNA-based RT-PCR techniques that are highly sensitive but as yet lack standardisation between laboratories. Since laboratories differ substantially in both their methodology and analysis strategy, interpretation and meaningful comparison of results between laboratories and from different studies is difficult or impossible. Moreover, and in contrast to CML, there are no generally accepted definitions of molecular response that could identify thresholds on which to base therapeutic decisions. Aims: To assess i) the variability of BCR-ABL quantification between laboratories with recognized expertise in bcr-abl analysis, ii) identify optimal laboratory methods and iii) to standardize analysis and interpretation of RT-PCR results for Ph+ALL, the EWALL and ESG-MRD-ALL consortia initiated a multinational project for quality assurance involving more than 30 laboratories from 14 countries worldwide. We here report the results of the first six m-BCR-ABL laboratory control rounds, performed between march 2008 and august 2011, that focussed on inter-intraassay variability of rt-PR techniques, comparison of housekeeping genes, plasmids, platforms and reagents. Methods: Serial dilutions of the BCR-ABL positive cell line Sup B15 with the BCR-ABL negative cell line Nalm 6 covering 5 logs were produced. In the 6 lab rounds, 1355 aliquots, each generated from with a total amount of 5 × 10E+06 cells in a final volume of 1 ml were produced, stabilized in TRIZOL or buffer RLT and frozen at −20°C until shipment or centralized isolation of RNA and reverse transcribed. Participants were asked to process the material using predefined procedures. Results: In the first QC round a major finding was the high variability in RNA-yield between laboratories despite using the same extraction method, with an up to 3 log difference in ABL copy numbers. In addition, there was rare false-positivity and false negativity (specifity 94.8%). With centrally produced cDNA the variability improved and there was a lower frequency of false-positivity reaching 100% specifity in QC 2. By using standardized assays, in 12 of 33 laboratories no conversion factor is needed, 20 of 33 laboratories are at the moment within one log difference when comparing the BCR-ABL/ABL ratios. Comparison of housekeeping genes (ABL/GUS) revealed non-linearity at high transcript levels resulting in an underestimation of intraindividual log reductions and may be relevant when assessing disease prognosis. The quantitative range was generally above 10E-04. Conclusion: Initiation of laboratory control rounds and implementation of standardized methodology improved the variability and reduced the frequency of false-positive results. Sensitivity of current techniques is still unsatisfactory for diseases with aggressive growth kinetics such as Ph*ALL. Further standardisation of technical and analytical methodology will provide the basis for defining levels of molecular remission and relapse and help to ensure comparability between laboratories and clinical trials at the European level. Disclosures: Goekbuget: Micromet: Consultancy. Ottmann:Novartis Corporation: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding.